H-fabp as early predictor of myocardial infarction

ABSTRACT

The present invention relates to a method for diagnosing myocardial infarction in a subject who suffers from acute coronary syndrome and has a cardiac troponin level which is detectable but lower than the level that is considered as being indicative for a myocardial infarction. Moreover, the present invention relates to a method for identifying a subject being susceptible to cardiac intervention, wherein the subject suffers from acute coronary syndrome and has a cardiac troponin level which is detectable but lower than a level that is considered as being indicative for a myocardial infarction. The methods of the present invention are based on the determination of H-FABP and, optionally, myoglobin in a sample of the subject and comparing the amount of H-FABP and, optionally, myoglobin to reference amounts.

RELATED APPLICATIONS

This application is a continuation of PCT/EP2008/056603 filed May 29,2008 and claims priority to EP 07116337.2 filed Sep. 13, 2007 and EP07109141.7 filed May 29, 2007.

FIELD OF THE INVENTION

The present invention relates to a method for diagnosing myocardialinfarction in a subject who suffers from acute coronary syndrome and hasa cardiac troponin level, which is detectable, but lower than the levelthat is considered as being indicative for a myocardial infarction.Moreover, the present invention relates to a method for identifying asubject being susceptible to cardiac intervention, wherein the subjectsuffers from acute coronary syndrome and has a cardiac troponin levelwhich is detectable, but lower than a level that is considered as beingindicative for a myocardial infarction. The methods of the presentinvention are based on the determination of heart-type fatty acidbinding protein (H-FABP) and, optionally, myoglobin in a sample of saidsubject and comparing the amount of heart-type fatty acid bindingprotein (H-FABP) and, optionally, myoglobin to at least one referenceamount. Also comprised by the present invention are kits or devices tocarry out the methods of the present invention.

BACKGROUND

An aim of modern medicine is to provide personalized or individualizedtreatment regimens. Those are treatment regimens which take into accounta patient's individual needs or risks. A particularly important risk isthe presence of cardiovascular complication, especially of an acutecardiovascular event. Cardiovascular complications belong to the leadingcauses of morbidity and mortality in the Western hemisphere. Forindividual treatment of a person who suffers from a cardiovascularcomplication, a reliable diagnosis has a significant impact on thesuccess of the treatment of said person. This is particularly importantfor patients showing symptoms of acute coronary syndrome (ACS).

Acute coronary syndrome refers to a constellation of clinical symptomscaused by acute myocardial ischemia. Patients with acute coronarysyndrome have a significantly increased risk of cardiac death and,therefore, need to be identified between the patients with nontraumaticchest symptoms (Morrow et al., National academy of clinical biochemistryguidelines: Clinical characteristics and utilization of biochemicalmarkers in acute coronary syndrome, 2007, Circulation; 115;356-375).Patients exhibiting symptoms of an acute cardiovascular event (e.g.,chest discomfort for more than 20 min) and presenting for emergencyevaluation are generally examined by electrocardiography. Moreover, ablood sample is obtained for determining the level of a cardiactroponin. Cardiac troponin, e.g., troponin T, is a biomarker formyocardial infarction (MI). The electrocardiogram (ECG) providesimportant information for the diagnosis. Particularly, if the ECG showselevated ST segments, a ST elevated myocardial infarction (STEM) isdiagnosed. If the ECG does not show elevated ST segments, a non STelevated MI (NSTEMI) is diagnosed when cardiac troponin is detected in asample of the respective patient. Patients without a diagnostic ECG andwith no detectable cardiac troponin are suspected to have unstableangina pectoris (UAP). Unstable angina and NSTEMI are considered to beclosely related conditions, sharing a similar clinical presentation.However, they differ in their severity. NSTEMI is distinguished fromunstable angina by ischemia causing irreversible myocardial damage whichis detectable by biomarkers of myocardial necrosis (Morrow et al., loc.cit.). In all described cases, thus STEMI, NSTEMI and UAP, the patientis treated according to the diagnosis.

Within the last two decades biomarkers like cardiac troponin have becomevaluable tools for the diagnosis of heart-associated diseases. Furthermarkers of heart-associated diseases are, e.g., NTproBNP, or creatinekinase isoenzyme MB (CK-MB). Recently, it was shown that thedetermination of myoglobin is a valuable tool for the diagnosis ofsubjects showing symptoms of ACS (Brogan G. X et al. Evaluation of a newrapid quantitative immunoassay for serum myoglobin versus CK-MB forruling out acute myocardial infarction in the emergency department.1994. Ann. Emerg. Med. 24(4):665-71). The determination of myoglobin isadvantageous because elevated concentrations can be determined shortlyafter the onset of symptoms (appr. 1 hour), and studies havedemonstrated its high sensitivity for detection of AMI within the firstfew hours of ACS. However, the use of myoglobin for the diagnosis ofmyocardial infarctions has some limitations. Although the concentrationof myoglobin rises quickly after the onset of symptoms, theconcentration drops quickly after approximately 8 to 16 hours (JamesMcCord J. at al. Ninety-Minute Exclusion of Acute Myocardial InfarctionBy Use of Quantitative Point-of-Care Testing of Myoglobin and troponinI. 2001. Circulation 104:1483; Morrow D. A. et al. National Academy ofClinical Biochemistry Laboratory Medicine Practice Guidelines: ClinicalCharacteristics and Utilization of Biochemical Markers in Acute CoronarySyndromes. 2007. Circulation, 115:356-375).

More recently, heart-type fatty acid binding protein (H-FABP) wassuggested as an early marker of myocardial infarction. Heart-type fattyacid-binding protein (H-FABP) is a low molecular weight cytoplasmicprotein and present abundantly in the myocardium. When the myocardium isinjured, as in the case of myocardial infarction, low molecular weightcytoplasmic proteins including H-FABP are released into the circulationand an elevated H-FABP level is detectable in a blood sample. (e.g.,Okamoto et al., Clin Chem Lab Med 38(3):231-8 (2000) Human heart-typecytoplasmic fatty acid-binding protein (H-FABP) for the diagnosis Ofacute myocardial infarction. Clinical evaluation of H-FABP in comparisonwith myoglobin and creatine kinase isoenzyme MB; O'Donoghue et al.,Circulation, 114;550-557 (2006) Prognostic Utility of Heart-Type FattyAcid Binding Protein in patients with acute coronary syndrome or Ruzgaret al., Heart Vessels, 21;209-314 (2006) The use of human heart-typefatty acid-binding protein as an early diagnostic marker of myocardialnecrosis in patients with acute coronary syndrome, and its comparisonwith troponin-T and its creatine kinase-myocardial band).

The finding that cardiac troponin, e.g., cardiac troponin T (TnT) orcardiac troponin I (TnI), is a marker of myocardial infarction hasrevolutionized the diagnosis and management of patients showing symptomsof ACS. Particularly, cardiac troponin T is a very specific marker ofdamage of the myocardium and, therefore, allows differentiating betweenUAP and MI in patients exhibiting symptoms of ACS. However, there arestill some problems related to the use of cardiac troponin as adiagnostic marker in patients with acute coronary syndrome. E.g, thecardiac troponin level is generally not elevated at the onset of thesymptoms of an acute coronary event. Generally, an elevated troponinlevel can be detected approximately 4 to 6 hours after the onset ofsymptoms of ACS. Thus, within the first 0 to 6 hours of an acutecardiovascular event, the use of troponin as a biomarker for thediagnosis of myocardial infarction causes a relatively high proportionof false negative results. Thus, a myocardial infarction might not berecognized by means of a cardiac troponin assay and this may result in apossibly inappropriate or delayed treatment. The introduction of a newgeneration of cardiac troponin tests, which are more sensitive thantroponin tests of the previous generations and, thus, can detect muchlower cardiac troponin levels, had enabled a more reliable and earlierdetection of elevated cardiac troponin levels. Thus, in case of amyocardial event a necrosis may be detected earlier. However, recentstudies brought evidence that, when using more sensitive troponin tests,cardiac troponin can also be reproducibly detected in patients with astable coronary heart disease who do not suffer from an acute event(unpublished data). Thus, if a person with a stable coronary heartdisease and an already elevated, but still low cardiac troponin levelshows symptoms of ACS, it is unclear whether the detectable elevatedcardiac troponin level is due to the acute event or due to the alreadyexisting coronary heart disease. This raises the possibility of anincorrect diagnosis, e.g., MI instead of UAP, resulting in a possiblyharmful, wrong and/or delayed treatment.

Therefore, there is a clear need for diagnostic and prognostic means andmethods allowing a reliable and quick diagnosis of. MI in a subject whoshows symptoms of an acute coronary syndrome and who has a cardiactroponin level which is close to the detection limit. The said means andmethods shall allow a diagnosis of said subject and shall allowidentifying a subject being susceptible to cardiac intervention, anappropriate treatment of said subject and shall avoid the drawbacks ofthe current techniques as laid out above.

Thus, the technical problem underlying the present invention must beseen as the provision of means and methods for complying with theaforementioned needs.

The technical problem is solved by the embodiments characterized in theclaims and herein below.

SUMMARY OF THE INVENTION

Accordingly, the present invention relates to a method for diagnosingmyocardial infarction in a subject who suffers from acute coronarysyndrome and has a cardiac troponin level which is detectable, but lowerthan the level that is considered as being indicative for a myocardialinfarction, comprising

-   -   (a) determining the amount of heart-type fatty acid binding        protein (H-FABP) in a sample of said subject,    -   (b) comparing the amount of H-FABP as determined in step a) to        at least one reference amount, and    -   (c) diagnosing myocardial infarction based on the information        obtained in steps a) and b)

In an embodiment of the aforementioned method of the present invention,additionally the amount of the myoglobin is determined in an additionalstep aa) in a sample of said subject and compared to at least onereference amount for myoglobin in an additional step bb). Accordingly,in step c′), myocardial infarction is diagnosed based on the determinedamounts of myoglobin and H-FABP and the comparison of the amount ofmyoglobin to at least one reference amount for myoglobin and thecomparison of the amount of H-FABP to at least one reference amount forH-FABP. Preferably, the determination of H-FABP is carried out beforethe determination of myoglobin, however it is also contemplated thedetermination is carried out in any order, i.e. simultaneously, or atfirst H-FABP and then myoglobin, or at first myoglobin and then H-FABP.

The method of the present invention, preferably, is an in vitro method.Moreover, it may comprise steps in addition to those explicitlymentioned above. For example, further steps may relate to samplepre-treatments or evaluation of the results obtained by the method. Themethod of the present invention may be also used for monitoring,confirmation, and subclassification of a diagnosis. The method may becarried out manually or assisted by automation. Preferably, step (a),(b) and/or (c) may in total or in part be assisted by automation, e.g.,by a suitable robotic and sensory equipment for the determination instep (a) or a computer-implemented comparison in step (b).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Receiver operating characteristic (ROC) curve for H-FABP.

ROC curve analysis was done to determine diagnostic accuracy bycalculation of diagnostic sensitivity vs. (1-specificity) for a givendiagnostic parameter (H-FABP) according clinical outcome (Non-MyocardialInfarction (MD-Converter vs. Myocardial Infarction (MI)-Converter).Included in this ROC curve are the data obtained for patients withstable coronary heart disease and ACS (see Examples). The cut-off scorefor the clinical outcome MI is 4950 pg/ml H-FABP. (ROC-AUC: Receiveroperating characteristic-area under curve; C. O. cut-off).

FIG. 2: Receiver operating characteristic (ROC) curve for myoglobin.

ROC curve analysis was done to determine diagnostic accuracy bycalculation of diagnostic sensitivity vs. (1-specificity) for a givendiagnostic parameter (myoglobin) according clinical outcome(Non-Myocardial Infarction (MI)-Converter vs. Myocardial. Infarction(MI)-Converter). Included in this ROC curve are the data obtained forpatients with stable coronary heart disease and ACS (see Examples). Thecut-off score for the clinical outcome MI is 61 ng/ml myoglobin.(ROC-AUC: Receiver operating characteristic-area under curve; C. O.cut-off).

DETAILED DESCRIPTION OF THE INVENTION

The term “diagnosing myocardial infarction” relates to assessing whetherin a subject as defined in the present invention (thus a subject, whosuffers from ACS and has a cardiac troponin level which is detectablebut lower than the level that is considered as being indicative for MI)a myocardial infarction has recently occurred or not, and, thus, whetherthe underlying cause for an ACS is a myocardial infarction or unstableangina pectoris. The term “myocardial infarction” is known by the personskilled in the art. The term relates to the irreversible necrosis of themyocardium as a result of prolonged ischemia. As it will be understoodby those skilled in the art, the diagnosis is usually not intended to becorrect for 100% of the subjects to be analyzed. The term, however,requires that the diagnosis will be valid for a statisticallysignificant portion of the subjects to be examined. Whether a portion isstatistically significant can be determined without further ado by theperson skilled in the art using various well known statistic evaluationtools, e.g., determination of confidence intervals, p-valuedetermination, Student's t-test, Mann-Whitney test, etc. Details arefound in Dowdy and Wearden, Statistics for Research, John Wiley & Sons,New York 1983. Preferred confidence intervals are at least 90%, at least95%, at least 97%, at least 98% or at least 99%. The p-values are,preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, theprobability envisaged by the present invention allows that the diagnosiswill be correct for at least 60%, at least 70%, at least 80%, or atleast 90% of the subjects of a given cohort.

The term “subject” as used herein relates to animals, preferablymammals, and, more preferably, humans.

The subject shall, preferably, show symptoms of ACS. The term “acutecoronary syndrome” (ACS) is understood by the person skilled in the art.The term relates to a constellation of clinical symptoms caused by acutemyocardial ischemia. The ischemia itself results from the disruption ofan atherosclerotic plaque in a coronary artery. Symptoms for ACS,preferably, are chest pain for more than 20 minutes, shortness ofbreath, nausea, vomiting and or sweating. Moreover, it is known that thechest pain frequently radiates to the left arm and the left angle of thejaw. Generally, these clinical symptoms, especially chest pain, occursuddenly; they may appear at rest or after minimal exertion. Moreover,in the context of the present invention, the term “acute coronarysyndrome” may also relate to suspected, assumed, or possible ACS, asthese terms are frequently used for patients which show symptomsconsistent with ACS, and but for which the diagnosis has not beenconclusively established (see Morrow, loc. cit.). ACS patients can showunstable angina pectoris (UAP) or these individuals can suffer from amyocardial infarction (MI). MI can be an ST-elevated MI (STEMI) or anon-ST-elevated MI (NSTEMI). MI is classified as belonging to coronaryheart diseases CHD and is preceded by other events also classified asbelonging to CHD, like unstable angina pectoris UAP. Symptomatic for UAPis chest pain which is relieved by sublingual administration ofnitroglycerine. UAP is caused by a partial occlusion of the coronaryvessels leading to hypoxemia and myocardial ischemia. In case, theocclusion is too severe or total, an irreversible myocardial necrosis(which is the pathological state underlying myocardial infarction)results. Generally, STEMI is diagnosed by electrocardiography, in casethe electrocardiogram (ECG) show ST-segment elevation. The determinationof a cardiac troponin level at least six hours after the onset ofsymptoms of ACS allows for differentiating UAP and NSTEMI. If thetroponin level is elevated (indicating myocardial damage) a NSTEMI isassumed. MI may occur without obvious symptoms, i.e. the subject doesnot show any discomfort, and the MI is not preceded by stable orunstable angina pectoris. The occurrence of an MI can be followed by aleft ventricular dysfunction (LVD).

It is particularly contemplated that the subject shall suffer fromcoronary heart disease (frequently also referred to as coronary arterydisease) prior to the ACS and already have detectable cardiac troponinlevels at the onset of symptoms of ACS (and thus also prior to the onsetof symptoms). Particularly, said subject shall have detectable levels ofa cardiac troponin but lower than the level that is considered as beingindicative for MI at the onset of symptoms of ACS. For said subject themethod of the present invention will be particularly advantageous since,in case of an ACS, it is unclear whether elevated levels of a cardiactroponin indicate an acute event (ongoing necrosis) or are due to analready existing coronary heart disease.

The term “cardiac troponin” refers to all troponin isoforms expressed incells of the heart and, preferably, the subendocardial cells. Theseisoforms are well characterized in the art as described, e.g., inAnderson 1995, Circulation Research, vol. 76, no. 4: 681-686 andFerrieres 1998, Clinical Chemistry, 44: 487-493. Preferably, cardiactroponin refers to troponin T and/or troponin I, and, most preferably,to troponin T. It is to be understood that isoforms of Troponins may bedetermined in the method of the present invention together, i.e.simultaneously or sequentially, or individually, i.e. withoutdetermining the other isoform at all. Amino acid sequences for humantroponin T and human troponin I are disclosed in Anderson, loc cit andFerrieres 1998, Clinical Chemistry, 44: 487-493.

The term “cardiac troponin” encompasses also variants of theaforementioned specific Troponins, i.e., preferably, of troponin T (TnT)or troponin I (TnI). Such variants have at least the same essentialbiological and immunological properties as the specific cardiacTroponins. In particular, they share the same essential biological andimmunological properties if they are detectable by the same specificassays referred to in this specification, e.g., by ELISA Assays usingpolyclonal or monoclonal antibodies specifically recognizing the saidcardiac Troponins. Moreover, it is to be understood that a variant asreferred to in accordance with the present invention shall have an aminoacid sequence which differs due to at least one amino acid substitution,deletion and/or addition wherein the amino acid sequence of the variantis still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%,97%, 98%, or 99% identical with the amino sequence of the specifictroponin. Variants may be allelic variants or any other species specifichomologs; paralogs, or orthologs. Moreover, the variants, referred toherein include fragments of the specific cardiac Troponins or theaforementioned types of variants as long as these fragments have theessential immunological and biological properties as referred to above.Such fragments may be, e.g., degradation products of the Troponins.Further included are variants which differ due to posttranslationalmodifications such as phosphorylation or myristylation.

The term “cardiac troponin level” as used herein relates to theconcentration of a cardiac troponin, preferably of TnT. Preferably, theternrrelates to the concentration of a cardiac troponin in a plasma orserum sample of a subject. The term “cardiac troponin level which isdetectable” may relate to any cardiac troponin level which differs fromzero and which is detectable by means and methods known the art, e.g.,by commercially available cardiac troponin assays. Preferably, the“cardiac troponin level which is detectable” relates to a concentrationthat is equal or larger than the lowest detection limit of the assayused for determining the troponin level. Preferably, the troponin levelwhich is detectable may relate to any concentration that is equal orlarger than 0.001 ng/ml, 0.005 ng/ml, 0.0075 ng/ml, more preferablyequal or larger than 0.01 ng/ml, or equal or larger than 0.002 ng/ml.Most preferably, the cardiac troponin level which is detectable relatesto any concentration that is equal or larger than 0.002 ng/ml. It is tobe understood that a detectable level (e.g., larger than 0.002 ng/ml) ofa cardiac troponin is considered to be an elevated level of said cardiactroponin since such levels are usually not detected in healthyindividuals. Moreover, an elevated level of a cardiac troponin indicatesnecrosis.

The term “troponin level which is considered as being indicative formyocardial infarction” relates to a commonly accepted troponinconcentration that indicates a myocardial infarction. Preferably, thelevel considered as being indicative for myocardial infarction relatesto a concentration that is above the 99th percentile concentration of asuitable reference population (cut-off score). This level is based upona recommendation that was made by a joint committee of the EuropeanSociety of Cardiology and the American College of Cardiology to avoidfalse positive results (The Joint European Society ofCardiology/American College of Cardiology Committee. Myocardialinfarction redefined—a consensus document of the joint European Societyof Cardiology/American College of Cardiology Committee for theRedefinition of Myocardial Infarction. J Am Coll Cardiol2000;36:959-969). The person skilled in the art knows how to select asuitable reference population and how to determine the 99th percentileconcentration. It is to be understood that this concentration may differbased on the used assay for determining the cardiac troponinconcentration and based on the selected reference population. Preferredcardiac troponin levels considered to be indicative for MI in thecontext of the present invention may be, but are not limited to 0.05ng/ml, 0.075 ng/ml, 0.09.9 ng/ml, 0.1 ng/ml, 0.2 ng/ml and 0.3 ng/ml.The most preferred cardiac troponin level considered to be indicativefor MI in the context of the present invention is 0.1 ng/ml.

In a preferred embodiment of the methods of the present invention, thetroponin level, particularly the troponin T level, in a subject whosuffers from acute coronary syndrome and has a cardiac troponin levelwhich is detectable, but lower than the level that is considered asbeing indicative for a myocardial infarction (as defined in thisapplication) ranges from 0.002 to 0.1 ng/ml (equal or larger than 0.002,but lower than 0.1 ng/ml).

Heart-type fatty acid binding protein, herein also referred to as H-FABPor heart fatty acid binding protein, is a small cytosolic protein thatfunctions as the principal transporter of long-chain fatty acids in thecardiomyocyte. H-FABP is present in the myocardium and it is generallythought to be released rapidly into the circulation in response tomyocardial injury. Several studies show that H-FABP is a earlybiochemical marker of myocardial infarction e.g., Okamoto et al., ClinChem Lab Med 38(3):231-8 (2000) Human heart-type cytoplasmic fattyacid-binding protein (H-FABP) for the diagnosis of acute myocardialinfarction. Clinical evaluation of H-FABP in comparison with myoglobinand creatine kinase isoenzyme MB; O'Donoghue et al., Circulation,114;550-557 (2006) Prognostic Utility of Heart-Type Fatty Acid BindingProtein in patients with acute coronary syndrome or Ruzgar et al., HeartVessels, 21;209-314 (2006) The use of human heart-type fattyacid-binding protein as an early diagnostic marker of myocardialnecrosis in patients with acute coronary syndrome, and its comparisonwith troponin T and its creatine kinase-myocardial band).). H-FABP iswell known in the art. Moreover, assays to determine the amount ofH-FABP are also well known.

Myoglobin is a cytoplasmic hemoprotein consisting of a singlepolypeptide chain of 154 amino acids and is almost exclusively expressedsolely in cardiac myocytes and oxidative skeletal muscle fibers. Likehemoglobin, myoglobin reversibly binds oxigen and thus may facilitateoxigen transport from red blood cells to mitochondria during periods ofincreased metabolic activity or serve as an oxigene reservoir duringhypoxic or anoxic conditions Ordway G. and Garry D. J., Myoglobin: anessential hemoprotein in striated muscle. 2004. Journal of ExperimentalBiology 207, 3441-3446 (2004). Myoglobin is well known in the art.Moreover, assays to determine the amount of myoglobin are also wellknown.

Myoglobin and H-FABP as used herein encompass also variants of myoglobinand H-FABP polypeptides, respectively. Such variants have at least thesame essential biological and immunological properties as the specificH-FABP and myoglobin polypeptides, respectively. In particular, theyshare the same essential biological and immunological properties if theyare detectable by the same specific assays referred to in thisspecification, e.g., by ELISA assays using polyclonal or monoclonalantibodies specifically recognizing the said H-FABP and myoglobinpolypeptides, respectively. Moreover, it is to be understood that avariant as referred to in accordance with the present invention shallhave an amino acid sequence which differs due to at least one amino acidsubstitution, deletion and/or addition wherein the amino acid sequenceof the variant is still, preferably, at least 50%, 60%, 70%, 80%, 85%,90%, 92%, 95%, 97%, 98%, or 99% identical with the amino sequence of thespecific H-FABP and myoglobin polypeptides, respectively. The degree ofidentity between two amino acid sequences can be determined byalgorithms well known in the art. Preferably, the degree of identity isto be determined by comparing two optimally aligned sequences over acomparison window, where the fragment of amino acid sequence in thecomparison window may comprise additions or deletions (e.g., gaps oroverhangs) as compared to the reference sequence (which does notcomprise additions or deletions) for optimal alignment. The percentageis calculated by determining the number of positions at which theidentical amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison andmultiplying the result by 100 to yield the percentage of sequenceidentity. Optimal alignment of sequences for comparison may be conductedby the local homology algorithm of Smith and Waterman Add. APL, Math.2:482 (1981), by the homology alignment algorithm of Needleman andWunsch J. Mol. Biol. 48:443 (1970), by the search for similarity methodof Pearson and Lipman Proc. Natl. Acad Sci. (USA) 85: 2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, BLAST,PASTA, and TFASTA in the Wisconsin Genetics. Software Package, GeneticsComputer Group (GCG), 575 Science Dr., Madison, Wis.), or by visualinspection. Given that two sequences have been identified forcomparison, GAP and BESTFIT are preferably employed to determine theiroptimal alignment and, thus, the degree of identity. Preferably, thedefault values of 5.00 for gap weight and 0.30 for gap weight length areused. Variants referred to above may be allelic variants or any otherspecies specific homologs, paralogs, or orthologs. Moreover, thevariants referred to herein include fragments of the specific H-FABP andmyoglobin polypeptides, respectively or the aforementioned types ofvariants as long as these fragments have the essential immunological andbiological properties as referred to above. Such fragments may be, e.g.,degradation products of the H-FABP and myoglobin polypeptides,respectively. Further, included are variants which differ due toposttranslational modifications such as phosphorylation ormyristylation.

The term “sample” refers to a sample of a body fluid, to a sample ofseparated cells or to a sample from a tissue or an organ. Samples ofbody fluids can be obtained by well known techniques and include,preferably, samples of blood, plasma, serum, or urine, more preferably,samples of blood, plasma or serum. Tissue or organ samples may beobtained from any tissue or organ by, e.g., biopsy. Separated cells maybe obtained from the body fluids or the tissues or organs by separatingtechniques such as centrifugation or cell sorting. Preferably, cell-,tissue- or organ samples are obtained from those cells, tissues ororgans which express or produce the peptides referred to herein.Preferably, the term “sample” refers to a plasma or serum sample, morepreferably to a serum sample.

The sample is obtained at an appropriate time-point which is known bythe skilled person. Preferably, the sample is obtained from a subjectaccording the present invention shortly, preferably not more than 2hours (and thus within 2 hours), and, more preferably, not more than 4hours, and more preferably, not more than 6 hours after the onset ofsymptoms of acute coronary syndrome. The method of the present inventionis particularly advantageous in cases the samples are obtained shortlyafter the onset of symptoms of ACS. In such cases it is unclear whethera detectable elevated cardiac troponin level is due to the acute eventor due to the already existing coronary heart disease.

Determining the amount of H-FABP, preferably human H-FABP or myoglobin,preferably human myoglobin or any other peptide or polypeptide orprotein referred to in this specification relates to measuring theamount or concentration, preferably semi-quantitatively orquantitatively. The terms polypeptide and protein are usedinterchangeable throughout this application. Measuring can be donedirectly or indirectly. Direct measuring relates to measuring the amountor concentration of the peptide or polypeptide based on a signal whichis obtained from the peptide or polypeptide itself and the intensity ofwhich directly correlates with the number of molecules, of the peptidepresent in the sample. Such a signal—sometimes referred to herein asintensity signal—may be obtained, e.g., by measuring an intensity valueof a specific physical or chemical property of the peptide orpolypeptide. Indirect measuring includes measuring of a signal obtainedfrom a secondary component (i.e. a component not being the peptide orpolypeptide itself) or a biological read out system, e.g., measurablecellular responses, ligands, labels, or enzymatic reaction products.

In accordance with the present invention, determining the amount of apeptide or polypeptide can be achieved by all known means fordetermining the amount of a peptide in a sample. Said means compriseimmunoassay devices and methods which may utilize labeled molecules invarious sandwich, competition, or other assay formats. Said assays willdevelop a signal which is indicative for the presence or absence of thepeptide or polypeptide. Moreover, the signal strength can, preferably,be correlated directly or indirectly (e.g., reverse-proportional) to theamount of polypeptide present in a sample. Further suitable methodscomprise measuring a physical or chemical property specific for thepeptide or polypeptide such as its precise molecular mass or NMRspectrum. Said methods comprise, preferably, biosensors, optical devicescoupled to immunoassays, biochips, analytical devices such asmass-spectrometers, NMR-analyzers, or chromatography devices. Further,methods include micro-plate ELISA-based methods, fully-automated orrobotic immunoassays (available for example on ELECSYS analyzers), CBA(an enzymatic Cobalt Binding Assay, available for example onRoche-Hitachi analyzers), and latex agglutination assays (available forexample on Roche-Hitachi analyzers).

Preferably, determining the amount of a peptide or polypeptide comprisesthe steps of (a) contacting a cell capable of eliciting a cellularresponse the intensity of which is indicative of the amount of thepeptide or polypeptide with the said peptide or polypeptide for anadequate period of time, (b) measuring the cellular response. Formeasuring cellular responses, the sample or processed sample is,preferably, added to a cell culture and an internal or external cellularresponse is measured. The cellular response may include the measurableexpression of a reporter gene or the secretion of a substance, e.g., apeptide, polypeptide, or a small molecule. The expression or substanceshall generate an intensity signal which correlates to the amount of thepeptide or polypeptide.

Also preferably, determining the amount of a peptide or polypeptidecomprises the step of measuring a specific intensity signal obtainablefrom the peptide or polypeptide in the sample. As described above, sucha signal may be the signal intensity observed at an m/z variablespecific for the peptide or polypeptide observed in mass spectra or aNMR spectrum specific for the peptide or polypeptide.

Determining the amount of a peptide or polypeptide may, preferably,comprises the steps of (a) contacting the peptide with a specificligand, (b) (optionally) removing non-bound ligand, (c) measuring theamount of bound ligand. The bound ligand will generate an intensitysignal. Binding according to the present invention includes bothcovalent and non-covalent binding. A ligand according to the presentinvention can be any compound, e.g., a peptide, polypeptide, nucleicacid, or small molecule, binding to the peptide or polypeptide describedherein. Preferred ligands include antibodies, nucleic acids, peptides orpolypeptides such as receptors or binding partners for the peptide orpolypeptide and fragments thereof comprising the binding domains for thepeptides, and aptamers, e.g., nucleic acid or peptide aptamers. Methodsto prepare such ligands are well-known in the art. For example,identification and production of suitable antibodies or aptamers is alsooffered by commercial suppliers. The person skilled in the art isfamiliar with methods to develop derivatives of such ligands with higheraffinity or specificity. For example, random mutations can be introducedinto the nucleic acids, peptides or polypeptides. These derivatives canthen be tested for binding according to screening procedures known inthe art, e.g., phage display. Antibodies as referred to herein includeboth polyclonal and monoclonal antibodies, as well as fragments thereof,such as Fv, Fab and F(ab)2 fragments that are capable of binding antigenor hapten. The present invention also includes single chain antibodiesand humanized hybrid antibodies wherein amino acid sequences of anon-human donor antibody exhibiting a desired antigen-specificity arecombined with sequences of a human acceptor antibody. The donorsequences will usually include at least the antigen-binding amino acidresidues of the donor but may comprise other structurally and/orfunctionally relevant amino acid residues of the donor antibody as well.Such hybrids can be prepared by several methods well known in the art.Preferably, the ligand or agent binds specifically to the peptide orpolypeptide. Specific binding according to the present invention meansthat the ligand or agent should not bind substantially to (“cross-react”with) another peptide, polypeptide or substance present in the sample tobe analyzed. Preferably, the specifically bound peptide or polypeptideshould be bound with at least 3 times higher, more preferably at least10 times higher and even more preferably at least 50 times higheraffinity than any other relevant peptide or polypeptide. Non-specificbinding may be tolerable, if it can still be distinguished and measuredunequivocally, e.g., according to its size on a Western Blot, or by itsrelatively higher abundance in the sample. Binding of the ligand can bemeasured by any method known in the art. Preferably, said method issemi-quantitative or quantitative. Suitable methods are described in thefollowing:

First, binding of a ligand may be measured directly, e.g., by NMR orsurface plasmon resonance.

Second, if the ligand also serves as a substrate of an enzymaticactivity of the peptide or polypeptide of interest, an enzymaticreaction product may be measured (e.g., the amount of a protease can bemeasured by measuring the amount of cleaved substrate, e.g., on aWestern Blot). Alternatively, the ligand may exhibit enzymaticproperties itself and the “ligand/peptide or polypeptide” complex or theligand which was bound by the peptide or polypeptide, respectively, maybe contacted with a suitable substrate allowing detection by thegeneration of an intensity signal. For measurement of enzymatic reactionproducts, preferably the amount of substrate is saturating. Thesubstrate may also be labeled with a detectable lable prior to thereaction. Preferably, the sample is contacted with the substrate for anadequate period of time. An adequate period of time refers to the timenecessary for a detectable, preferably measurable, amount of product tobe produced. Instead of measuring the amount of product, the timenecessary for appearance of a given (e.g., detectable) amount of productcan be measured.

Third, the ligand may be coupled covalently or non-covalently to a labelallowing detection and measurement of the ligand. Labeling may be doneby direct or indirect methods. Direct labeling involves coupling of thelabel directly (covalently or non-covalently) to the ligand. Indirectlabeling involves binding (covalently or non-covalently) of a secondaryligand to the first ligand. The secondary ligand should specificallybind to the first ligand. Said secondary ligand may be coupled with asuitable label and/or be the target (receptor) of tertiary ligandbinding to the secondary ligand. The use of secondary, tertiary or evenhigher order ligands is often used to increase the signal. Suitablesecondary and higher order ligands may include antibodies, secondaryantibodies, and the well-known streptavidin-biotin system (VectorLaboratories, Inc.). The ligand or substrate may also be “tagged” withone or more tags as known in the art. Such tags may then be targets forhigher order ligands. Suitable tags include biotin, digoxygenin,His-Tag, Glutathion-S-Transferase, FLAG, GFP, myc-tag, influenza A virushaemagglutinin (HA), maltose binding protein, and the like. In the caseof a peptide or polypeptide, the tag is preferably at the N-terminusand/or C-terminus. Suitable labels are any labels detectable by anappropriate detection method. Typical labels include gold particles,latex beads, acridan ester, luminol, ruthenium, enzymatically activelabels, radioactive labels, magnetic labels (“e.g., magnetic beads”,including paramagnetic and superparamagnetic labels), and fluorescentlabels. Enzymatically active labels include e.g., horseradishperoxidase, alkaline phosphatase, beta-Galactosidase, Luciferase, andderivatives thereof. Suitable substrates for detection includedi-amino-benzidine (DAB), 3,3′-5,5′-tetramethylbenzidine, NBT-BCIP(4-nitro blue tetrazolium chloride and5-bromo-4-chloro-3-indolyl-phosphate, available as ready-made stocksolution from Roche Diagnostics), CDP-Star (Amersham Biosciences). ECF(Amersham Biosciences). A suitable enzyme-substrate combination mayresult in a colored reaction product, fluorescence or chemiluminescence,which can be measured according to methods known in the art (e.g., usinga light-sensitive film or a suitable camera system). As for measuringthe enyzmatic reaction, the criteria given above apply analogously.Typical fluorescent labels include fluorescent proteins (such as GFP andits derivatives), Cy3, Cy5, Texas Red, Fluorescein, and the Alexa dyes(e.g., Alexa 568). Further fluorescent labels are available e.g., fromMolecular Probes (Oregon). Also the use of quantum dots as fluorescentlabels is contemplated. Typical radioactive labels include 35S, 125I,32P, 33P and the like. A radioactive label can be detected by any methodknown and appropriate, e.g., a light-sensitive film or a phosphorimager. Suitable measurement methods according the present inventionalso include precipitation (particularly immunoprecipitation),electrochemiluminescence (electro-generated chemiluminescence), RIA(radioimmunoassay), ELISA (enzyme-linked immunosorbent assay), sandwichenzyme immune tests, electrochemiluminescence sandwich immunoassays(ECLIA), dissociation-enhanced lanthanide fluoro immuno assay (DELFIA),scintillation, proximity assay (SPA), turbidimetry, nephelometry,latex-enhanced turbidimetry or nephelometry, or solid phase immunetests. Further methods known in the art (such as gel electrophoresis, 2Dgel electrophoresis, SDS polyacrylamide gel electrophoresis (SDS-PAGE),Western Blotting, and mass spectrometry), can be used alone or incombination with labelling or other detection methods as describedabove.

The amount of a peptide or polypeptide may be, also preferably,determined as follows: (a) contacting a solid support comprising aligand for the peptide or polypeptide as specified above with a samplecomprising the peptide or polypeptide and (b) measuring the amountpeptide or polypeptide which is bound to the support. The ligand,preferably chosen from the group consisting of nucleic acids, peptides,polypeptides, antibodies and aptamers, is preferably present on a solidsupport in immobilized form. Materials for manufacturing solid supportsare well known in the art and include, inter alia, commerciallyavailable column materials, polystyrene beads, latex beads, magneticbeads, colloid metal particles, glass and/or silicon chips and surfaces,nitrocellulose strips, membranes, sheets, duracytes, wells and walls ofreaction trays, plastic tubes etc. The ligand or agent may be bound tomany different carriers. Examples of well-known carriers include glass,polystyrene, polyvinyl chloride, polypropylene, polyethylene,polycarbonate, dextran, nylon, amyloses, natural and modifiedcelluloses, polyacrylamides, agaroses, and magnetite. The nature of thecarrier can be either soluble or insoluble for the purposes of theinvention. Suitable methods for fixing/immobilizing said ligand are wellknown and include, but are not limited to ionic, hydrophobic, covalentinteractions and the like. It is also contemplated to use “suspensionarrays” as arrays according to the present invention (Nolan 2002, TrendsBiotechnol. 20(1):9-12). In such suspension arrays, the carrier, e.g., amicrobead or microsphere, is present in suspension. The array consistsof different microbeads or microspheres, possibly labeled, carryingdifferent ligands. Methods of producing such arrays, for example basedon solid-phase chemistry and photo-labile protective groups, aregenerally known (U.S. Pat. No. 5,744,305).

Preferably, the amount of the amount of H-FABP and the amount ofmyoglobin (if myoglobin is measured) are determined in a blood sampleobtained from a subject as defined in the present invention. Preferably,such a determination is done by ELISA. Such a determination by ELISA canbe done, e.g., by using the HBT ELISA Test Kit for human heart typefatty acid binding protein (HyCult Biotechnology, Uden, The Netherlands)for the determination of the amount of H-FABP and by using theTina-Quant Myoglobin Test System (Roche Diagnostics) for thedetermination of the amount of myoglobin, respectively.

The term “amount” as used herein encompasses the absolute amount (e.g.,of H-FABP or myoglobin), the relative amount or concentration (e.g., ofH-FABP or myoglobin) as well as any value or parameter which correlatesthereto. Such values or parameters comprise intensity signal values fromall specific physical or chemical properties obtained from the saidpeptides by direct measurements, e.g., intensity values in mass spectraor NMR spectra. Moreover, encompassed are all values or parameters whichare obtained by indirect measurements specified elsewhere in thisdescription, e.g., expression levels determined from biological read outsystems in response to the peptides or intensity signals obtained fromspecifically bound ligands. It is to be understood that valuescorrelating to the aforementioned amounts or parameters can also beobtained by all standard mathematical operations.

The term “comparing” as used herein encompasses comparing the amount ofthe peptide, polypeptide, protein comprised by the sample to be analyzedwith an amount of a suitable reference source specified elsewhere inthis description. It is to be understood that comparing as used hereinrefers to a comparison of corresponding parameters or values, e.g., anabsolute amount is compared to an absolute reference amount while aconcentration is compared to a reference concentration or an intensitysignal obtained from a test sample is compared to the same type ofintensity signal of a reference sample. The comparison referred to instep (b) of the method of the present invention may be carried outmanually or computer assisted. For a computer assisted comparison, thevalue of the determined amount may be compared to values correspondingto suitable references which are stored in a database by a computerprogram. The computer program may further evaluate the result of thecomparison, i.e. automatically provide the desired assessment in asuitable output format. Based on the comparison of the amount(s)determined in step a) to suitable reference amount(s), it is possible todiagnose MI in said subject. It is to be understood that an amount ofH-FABP as determined in step (a) of the methods of the presentsinvention is compared in step (b) to a reference amount for H-FABP asspecified elsewhere in this application and that an amount of myoglobinis compared to a reference amount for myoglobin.

Accordingly, the term “reference amount” as used herein either refers toan amount which allows for ruling in a recent occurrence of MI or to anamount which allows for ruling out a recent occurrence of MI in asubject who suffers from acute coronary syndrome and has a cardiactroponin level which is detectable but lower than the level that isconsidered as being indicative for a myocardial infarction (thus in asubject as defined in the present invention). Recent occurrence in thiscontext means that the MI has occurred, preferably, within a 6 hourperiod, more preferably within a 4 hour period, and most preferablywithin a 2 hour period before the sample was obtained from said subject.Preferably, a reference amount for ruling in MI may be derived fromsubject as defined in the present invention known to have suffered froma MI, preferably with within a 6 hour period, more preferably within a 4hour period and most preferably within a 2 hour period before the samplewas obtained. A reference amount for ruling out the recent occurrence ofMI may be derived from a subject as defined in the present inventionknown to not have suffered from MI. Furthermore, a reference amount forruling out the recent occurrence of a MI may be derived from a subjectwith stable coronary heart disease who has a low but detectable cardiactroponin level (as specified above) and who did not suffer from MI. Anamount of H-FABP and an amount of myoglobin (in cases myoglobin isdetermined) in a subject as defined in the present invention of largerthan the reference amount for ruling in the occurrence of MT shall beindicative for a recent occurrence of MI in said subject (and thus thatthe cause for the ACS is a MI). An amount of H-FABP and, if myoglobin isdetermined, an amount of myoglobin in a subject as defined in thepresent invention lower than the reference amount for ruling out theoccurrence of MI shall be an indicator that a MI infarction has notoccurred recently, thus said subject, preferably, suffers from UAP (andthus is an indicator that the cause for the ACS is UAP). It is to beunderstood in the context of the present invention that subjects asdefined in the present invention whose H-FABP amount is between theabove mentioned reference amounts (the reference amount for ruling inthe recent occurrence of MI and the reference amount for ruling out therecent occurrence of MI) may be required to be diagnosed again.Preferably, this may be also done for the rare case in which both theamount of myoglobin and H-FABP are determined and in which the amountsdo not correspond, e.g., one amount is larger (or lower) than therespective reference amount, whereas the other amount is not larger (orlower) than the respective reference amount. The new diagnosis,preferably, is done by determining the amount of the marker(s) and in anew, thus, later obtained sample of said subject. The new sample may beobtained from a subject; preferably, 1 hour, 2 hours, 3 hours afterobtaining the first sample. In addition, myoglobin may be determined insaid new sample. After obtaining the sample, the amount of H-FABP and ofmyoglobin can be determined in said new sample. Then, the thus obtainedresult(s) may be compared to reference amounts (as described elsewherein this application). Preferably, the amount of a cardiac troponin isalso determined in the sample and used for the diagnosis. Preferably, anamount of cardiac troponin T of larger than 0.1 ng/ml six hours aftershowing symptoms of ACS is indicative for MI.

The person skilled in the art knows how to determine a reference amount.It will be appreciated that the reference amount may also be chosenaccording to the desired sensitivity or a specificity of the diagnosis.Therefore, the reference amount may be chosen by the persons skilled inthe art according to the desired sensitivity and specificity. Means fordetermining suitable reference amounts are known to the persons skilledin the art, e. g. a reference amount can be determined fromReceiver-Operator-Curves (ROC) according to clinical studies.

A reference amount for H-FABP for ruling in the recent occurrence of MIin a subject as defined in the present invention defining a thresholdamount for H-FABP is 4950 pg/ml, 5550 pg/ml or 6000 pg/ml or, morepreferably, 5700 pg/ml.

An amount of H-FABP larger than the reference amount for H-FABP forruling in the recent occurrence of MI is, more preferably, indicativefor the recent occurrence of a MI, particularly a NSTEMI.

If in addition to H-FABP an amount of myoglobin is determined in asample of a subject as defined in the present invention and compared toa reference amount, a reference amount for myoglobin for ruling in therecent occurrence of MI in a subject as defined in the present inventiondefining a threshold amount for myoglobin is 64 ng/ml, or 69 ng/ml and,more preferably, 77 ng/ml.

An amount of myoglobin larger than the reference amount for myoglobinfor ruling in the recent occurrence of MI is, more preferably,indicative for the recent occurrence of a MI, particularly a NSTEMI,provided that also the amount of H-FABP in a sample of a subject is alsolarger than the reference amount for H-FABP for ruling in the recentoccurrence of MI.

A reference amount for H-FABP for ruling out the recent occurrence of MIin a subject according to the present invention defining a thresholdamount for H-FABP is 2100 pg/ml or 2300 pg/ml or, more preferably, 2500pg/ml.

An amount of H-FABP lower than the reference amount for H-FABP forruling out the recent occurrence is, more preferably, indicative that amyocardial infarction did not occur in a subject as defined in thepresent invention. Preferably, as a consequence the occurrence of a MIcan be ruled out and, e.g., a UAP can be assumed.

If in addition to H-FABP an amount of myoglobin is determined in asample of a subject as defined in the present invention and compared toa reference amount, a reference amount for myoglobin for ruling out therecent occurrence of MI in a subject according to the present inventiondefining a threshold amount for myoglobin is 28 ng/ml, or 61 ng/ml, or,more preferably, 55 ng/ml.

An amount of myoglobin lower than the reference amount for myoglobin forruling out the recent occurrence of MI is, more preferably, indicativethat a myocardial infarction did not occur in a subject as defined inthe present invention, provided that that also the amount of H-FABP in asample of a subject is also lower than the reference amount for H-FABPfor ruling out the recent occurrence of MI. Preferably, as a consequencethe occurrence of a MI can be ruled out and, e.g., a UAP can be assumed.

The term “at least one reference amount” means one or more than onereference amount, e.g., two reference amounts, e.g., the referenceamount for ruling in the recent occurrence of MI and the referenceamount for ruling out the recent occurrence of MI.

Advantageously, it has been found in the studies underlying the presentinvention that determining the amount of H-FABP in a subject as definedin the present invention (thus a subject suffering from acute coronarysyndrome and having a troponin level which is detectable, but lower thanthe level considered as being indicative for MI) and comparing thedetermined amount to at least one reference amount is required fordiagnosing MI in said subject. The aforementioned method is morereliable than those of the prior art since it has been found that H-FABPis required to assess the occurrence of a MI in case of low, butdetectable cardiac troponin T levels shortly after the onset of symptomsof an ACS or assumed ACS. In the studies underlying the presentinvention, H-FABP levels and TnT levels were determined in patientsshowing symptoms of ACS (within the first 2 hours after the onset ofsymptoms). The TnT level was determined by using a highly sensitivetroponin T assay with a detection limit of 0.002 ng/ml. Controlexperiments were carried out in which the amounts of TnT an H-FABP inpatients with stable coronary disease were determined (i.e. in patientswithout an apparent acute event). These experiments showed that TnT canalso be detected in subjects with stable coronary disease. A ReceiverOperating Characteristic (ROC) curve analysis including the data of theaforementioned studies further illustrated that H-FABP is a strongbiochemical marker for myocardial infarction (FIG. 1). Particularly, anH-FABP amount in a subject as defined in the present invention of largerthan 5700 pg/ml indicates a recent occurrence of MI (rule in), whereasan amount of less than 2500 pg/ml indicates that a MI did not occurrecently (rule out). Moreover, the sensitivity and specificity of adiagnosis based on the determination of H-FABP in a sample of a subjectas defined in the present invention is even more increased whenadditionally the amount of myoglobin in a sample of a subject isdetermined and compared to reference amounts for myoglobin.Particularly, a myoglobin amount in a subject as defined in the presentinvention of larger than 77 ng/ml indicates a recent occurrence of MI(rule in), whereas an amount of less than 55 ng/ml indicates that a MIdid not occur recently (rule out), FIG. 2. Thanks to this aspect of thepresent invention, a diagnosis for patients with ACS or assumed ACS witha low but detectable cardiac troponin level (detectable but lower thanthe level considered to be indicative for MI) can be more reliablyperformed. The findings of the study underlying the present inventionmay be particularly advantageous for the diagnosis of a) subjects whoalready have a low, but detectable, thus elevated levels of a cardiactroponin due to an already existing coronary heart disease and thenshows symptoms of ACS and b) of subjects with ACS with a low, butdetectable cardiac troponin level at the time at which the sample forthe determination of the cardiac troponin level was obtained (e.g.,because the sample was obtained too early) but in which a MI hasrecently occurred. In both cases, a) and b), the determination of H-FABPwill be a valuable tool for the diagnosis, particularly fordifferentiating between UAP and MI. After diagnosis, the subject can betreated accordingly. Without the determination of H-FABP, the diagnosismight be incorrect resulting in a putatively wrong, harmful and/ordelayed treatment of the mentioned subjects. Moreover, the sensitivityand specificity of diagnosis can be even more enhanced when alsomyoglobin is determined.

In a preferred embodiment of the method of the present invention themethod allows differentiating between myocardial infarction (MI) andunstable angina pectoris (UAP) in a subject who suffers from acutecoronary syndrome and has a cardiac troponin level which is detectable,but lower than the level that is considered as being indicative for amyocardial infarction (MI).

Accordingly, the method of the present invention is, in an embodiment, amethod for differentiating between myocardial infarction and unstableangina pectoris in a subject who suffers from acute coronary syndromeand has a cardiac troponin level which is detectable, but lower than thelevel that is considered as being indicative for a myocardial infarction(MI), comprising

-   -   a) determining the amount of heart type fatty acid binding        protein (H-FABP) in a sample of said subject, and    -   b) comparing the amount of H-FABP determined in step a) to at        least one reference amount, and    -   c) differentiating between myocardial infarction and unstable        angina pectoris based on the information obtained in steps a)        and b).

It is particularly contemplated that the subject shall have low, butdetectable levels of a cardiac troponin (thus, detectable but lower thanthe level that is considered as being indicative for MI) at the onset ofsymptoms of ACS (and, thus, shall have said have said levels prior tothe ACS). Preferably, said detectable levels are due to a coronary heartdisease. As mentioned above, in case of ACS it is difficult to decidewhether the detectable levels are due to the existing coronary heartdisease or due to the present ACS.

Therefore, in a preferred embodiment of the method of the presentinvention the method is for diagnosing myocardial infarction in asubject who suffers from acute coronary syndrome and has a cardiactroponin level which is detectable, but lower than the level that isconsidered as being indicative for a myocardial infarction (MI), and whohad low, but detectable cardiac troponin levels already at the onset ofsymptoms of ACS.

Accordingly, the method of the present invention is, in an embodiment, amethod for diagnosing myocardial infarction in a subject who suffersfrom acute coronary syndrome and has a cardiac troponin level which isdetectable, but lower than the level that is considered as beingindicative for a myocardial infarction (MI), and who had low, butdetectable cardiac troponin levels already at the onset of symptoms ofACS, comprising the steps

-   -   a) determining the amount of heart type fatty acid binding        protein (H-FABP) in a sample of said subject, and    -   b) comparing the amount of H-FABP determined in step a) to at        least one reference amount, and    -   c) diagnosing myocardial infarction based on the information        obtained in steps a) and b).

In accordance with the foregoing, the present invention can also be usedto rule in/rule out MI in a subject suffering from ACS.

Therefore, in another preferred embodiment of the method of the presentinvention the method is for ruling out myocardial infarction in asubject who suffers from acute coronary syndrome and has a cardiactroponin level which is detectable, but lower than the level that isconsidered as being indicative for a myocardial infarction (MI), and whohad low, but detectable cardiac troponin levels already at the onset ofsymptoms of ACS (and, thus, also shortly before the onset of symptoms).Preferably, MI is ruled out if the level of H-FABP is lower than thereference amount for ruling out MI. If both H-FABP and myoglobin aredetermined, MI is ruled out if, preferably, if both markers are lowerthan the respective reference amount for ruling out MI.

Accordingly, the method of the present invention is, in an embodiment, amethod for ruling out myocardial infarction in a subject who suffersfrom acute coronary syndrome and has a cardiac troponin level which isdetectable, but lower than the level that is considered as beingindicative for a myocardial infarction (MI), and who, preferably, hadlow, but detectable cardiac troponin levels already at the onset ofsymptoms of ACS comprising

-   -   a) determining the amount of heart type fatty acid binding        protein (H-FABP) in a sample of said subject, and    -   b) comparing the amount of H-FABP determined in step a) to a        reference amount for ruling out myocardial infarction, and    -   c) ruling out myocardial infarction, if the amount of H-FABP is        lower than the reference amount for ruling out myocardial        infarction.

In another preferred embodiment of the method of the present inventionthe method is for ruling in myocardial infarction in a subject whosuffers from acute coronary syndrome and has a cardiac troponin levelwhich is detectable, but lower than the level that is considered asbeing indicative for a myocardial infarction (MI), and who, preferably,had low, but detectable cardiac troponin levels already at the onset ofsymptoms of ACS. Preferably, MI is ruled in if the level of H-FABP islarger than the reference amount for ruling in MI. If both H-FABP andmyoglobin are determined, MI is ruled in, preferably, if both markersare larger than the respective reference amount for ruling in MI.

Explanations of the terms used for aforementioned methods can be foundelsewhere herein. Preferably, they also comprise the determination ofthe amount of myoglobin and comparing said amount to a reference amount.

It is to be understood that according to the method of the presentinvention described herein above and below, the amount of H-FABP and,preferably, also myoglobin, or means for the determination thereof canbe used for the manufacture of a diagnostic composition for diagnosingMI in a subject suffering from acute coronary syndrome and having acardiac troponin level which is detectable but lower than the levelconsidered as being indicative a myocardial infarction.

The present invention further relates to a method for identifying asubject being susceptible to cardiac intervention, whereby the subjectsuffers from acute coronary syndrome and has a cardiac troponin levelwhich is detectable, but lower than a level that is considered as beingindicative for a myocardial infarction, comprising carrying out thesteps a) and b) and, optionally, steps aa) and bb) as laid out in anyone of the aforementioned methods, and c) identifying a subject beingsusceptible to cardiac intervention.

Thanks to the aforementioned method, a risk/success stratification canbe easily performed before subjecting a patient to a cardiacintervention. In case the patient turns out to be not susceptible for acardiac intervention, said dangerous, time and/or cost intensive therapycan be avoided. Thus, besides preventing a subject from the adverse andsevere side effects accompanying a cardiac intervention, the method ofthe present invention will be beneficial for the health system in thatresources will be saved.

It is to be understood in the context of the aforementioned method ofthe present invention that a subject diagnosed to suffer from MI, thus asubject in which a MI has recently occurred, is susceptible to cardiacintervention.

The term “identifying” as used herein means assessing whether a subjectwill be susceptible for a cardiac intervention or not. As will beunderstood by those skilled in the art, such an assessment is usuallynot intended to be correct for all (i.e. 100%) of the subjects to beidentified. The term, however, requires that a statistically significantportion of subjects can be identified (e.g., a cohort in a cohortstudy). Whether a portion is statistically significant can be determinedwithout further ado by the person skilled in the art using various wellknown statistic evaluation tools, e.g., determination of confidenceintervals, p-value determination, Student's t-test, Mann-Whitney testetc. Details are found in Dowdy and Wearden, Statistics for Research,John Wiley & Sons, New York 1983. Preferred confidence intervals are atleast 90%, at least 95%, at least 97%, at least 98% or at least 99%. Thep-values are, preferably, 0.1, 0.05, 0.01, 0.005, or 0.0001. Morepreferably, at least 60%, at, least 70%, at least 80% or at least 90% ofthe subjects of a population can be properly identified by the method ofthe present invention.

The term “cardiac intervention” encompasses those treatment regimens forMI considered appropriate by the person skilled in the art. The termcomprises interventions by surgery, microsurgery, other invasivetherapies affecting the cardiovascular system and, preferably, theheart, as well as conservative (non-surgery) methods of treatment.Conservative methods are known in the art and includenon-pharmacological methods and pharmacological methods. Pharmacologicalmethods relate to the administration of pharmaceuticals (such asheparin, acetylsalicylic acid, clopidogrel) in therapeutic effectiveamounts. Preferably, cardiac interventions as used herein are treatmentregimens which aim to restore the proper oxygen supply of the heart.This is, preferably, achieved by restoring the blood flow throughout theblood vessels supporting the heart, i.e. the coronary blood vessels.Those blood vessels may be impaired due to, e.g., thrombotic oratherosclerotic plaques. Accordingly, cardiac interventions shall,preferably, comprise a destruction and/or removal of such plaques and arestoration of the vessel, if necessary. Preferred cardiac interventionsin accordance with the present invention are selected from the groupconsisting of percutaneous coronary angioplasty, percutaneoustransluminal coronary balloon angioplasty, laser angioplasty, coronarystent implantation, bypass implantation or intraluminal techniquesaiming to restore blood flow, vessel patency, stabilize plaque, and/orreduce intracoronary thrombus load.

Moreover, the present invention relates to a method of deciding on thepossible treatment of a subject who suffers from acute coronary syndromeand has a cardiac troponin level which is detectable, but lower than thelevel that is considered as being indicative for a myocardialinfarction, comprising

-   -   a) determining the amount of H-FABP in a sample of said subject,        and    -   b) comparing the amount of H-FABP determined in step a) to at        least one reference amount, and    -   c) recommending the initiation of a cardiac intervention or        refraining from the cardiac intervention, based on the        information obtained in steps a) and b).

In an embodiment of the aforementioned method of the present invention,additionally the amount of the myoglobin is determined step a) in asample of a subject and compared to at least one reference amount formyoglobin in step b). Accordingly, in the step c) the recommendation ofthe initiation or cardiac intervention and or the refraining from acardiac intervention is done based on the determined amounts of H-FABPand myoglobin, the comparison of the amount of myoglobin to at least onereference amount for myoglobin and the comparison of the amount ofH-FABP to at least one reference amount for H-FABP.

Moreover, encompassed by the present invention is a kit or device forcarrying out the methods of the present invention comprising means fordetermining the amount of H-FABP and in a sample of a subject and meansfor comparing said amount to at least one reference amount. Preferably,the kit or device also comprises means for determining the amount ofmyoglobin in a sample of a subject and means for comparing said amountto at least one reference amount.

The term “kit” as used herein refers to a collection of theaforementioned means, preferably, provided separately or within a singlecontainer. The kit may in addition comprise means for determining theamount of a cardiac troponin. Preferably, the kit may additionallycomprise a user's manual for interpreting the results of anymeasurement(s) with respect to diagnosing MI in a subject as defined inthe present invention. Particularly, such manual may include informationabout what determined amounts corresponds to what kind of diagnosis.This is outlined in detail elsewhere in this specification.Additionally, such user's manual may provide instructions aboutcorrectly using the components of the kit for determining the amount ofthe respective biomarkers.

The term “device” as used herein relates to a system of means comprisingat least the aforementioned means operatively linked to each other as toallow the diagnosis of MI or the identification of a subject beingsusceptible to cardiac intervention. The device invention may inaddition comprise means for determining the amount of a cardiactroponin. Preferred means for determining the amount or myoglobin andH-FABP and means for carrying out the comparison are disclosed above inconnection with the method of the invention. How to link the means in anoperating manner will depend on the type of means included into thedevice. For example, where means for automatically determining theamount of the peptides are applied, the data obtained by saidautomatically operating means can be processed by, e.g., a computerprogram in order to obtain the desired results. Preferably, the meansare comprised by a single device in such a case. Said device mayaccordingly include an analyzing unit for the measurement of the amountof the peptides or polypeptides in an applied sample and a computer unitfor processing the resulting data for the evaluation. Alternatively,where means such as test stripes are used for determining the amount ofthe peptides or polypeptides, the means for comparison may comprisecontrol stripes or tables allocating the determined amount to areference amount. The test stripes are, preferably, coupled to a ligandwhich specifically binds to the peptides or polypeptides referred toherein. The strip or device, preferably, comprises means for detectionof the binding of said peptides or polypeptides to the said ligand.Preferred means for detection are disclosed in connection withembodiments relating to the method of the invention above. In such acase, the means are operatively linked in that the user of the systembrings together the result of the determination of the amount and thediagnostic or prognostic value thereof due to the instructions andinterpretations given in a manual. The means may appear as separatedevices in such an embodiment and are, preferably, packaged together asa kit. The person skilled in the art will realize how to link the meanswithout further ado. Preferred devices are those which can be appliedwithout the particular knowledge of a specialized clinician, e.g., teststripes or electronic devices which merely require loading with asample. The results may be given as output of raw data which needinterpretation by the Clinician. Preferably, the output of the deviceis, however, processed, i.e. evaluated, raw data the interpretation ofwhich does not require a clinician. Further preferred devices comprisethe analyzing units/devices (e.g., biosensors, arrays, solid supportscoupled to ligands specifically recognizing the myoglobin or H-FABP,Plasmon surface resonance devices, NMR spectrometers, mass-spectrometersetc.) or evaluation units/devices referred to above in accordance withthe method of the invention.

The present invention also relates to the use of H-FABP and, preferablyin addition, myoglobin and/or means for determining the amount of H-FABPand, preferably in addition, myoglobin and/or means for comparing theamount of H-FABP and, preferably in addition, myoglobin to at least onereference amount for the manufacture of a diagnostic composition fordiagnosing myocardial infarction in a subject.

All references cited in this specification are herewith incorporated byreference with respect to their entire disclosure content and thedisclosure content specifically mentioned in this specification.

The following Examples shall merely illustrate the invention. They shallnot be construed, whatsoever, to limit the scope of the invention.

Example 1 Myoglobin, H-FABP and Troponin T in Patients with AcuteCoronary Syndrome

69 patients showing characteristic symptoms of ACS (e.g., chest pain)were examined. Blood samples were obtained within the first two hoursafter the onset of symptoms. For the diagnosis of a ST-elevated MIpatients were examined by electrocardiography. Additionally, thetroponin T concentration was determined with a troponin T assay with adetection limit of 0.01 ng/ml. Further blood samples were obtained frompatients for whom a diagnosis STEMI or NSTEMI could not be made (TnTconcentration larger 0.01 but lower than 0.1 ng/ml, thus, levels whichindicate necrosis). A troponin T level of larger than 0.1 ng/ml in asample that was obtained at least 6 hours after the onset of symptomswas considered as being indicative for the recent occurrence MI(MI-converter), otherwise UAP was diagnosed (Non-MI-converter).

In a later analysis, the TnT, myoglobin and H-FABP concentration insamples from patients for whom a diagnosis of STEMI or NSTEMI could notbe made (TnT in a first sample detectable, larger 0.01 ng/ml, but lower0.1 ng/ml) were determined by using a highly-sensitive TnT assay with adetection limit of 0.002 ng/ml, TINA-QUANT Myoglobin Test System (RocheDiagnostics GmbH) and a H-FABP ELISA Test Kit, respectively (HBT ELISATest kit for human heart type fatty acid binding protein; HyCultBiotechnology, Uden, The Netherlands). The results are shown in thefollowing table.

TABLE troponin T, H-FABP and myoglobin concentrations (Medians) inpatients with acute coronary syndrome (MI Converter and Non-MIConverter). Heart type- High-Sensitive Fatty Acid troponin T BindingProtein Myoglobin [pg/ml] [pg/ml] [ng/ml] Non-MI- MI Non-MI MI Non-MI MIN 42 27 42 27 42 27 Median 4.35 22.75 2896.39 8114.01 36.89 76.76

Example 2 Myoglobin, H-FABP and Troponin T in Patients with StableCoronary Heart Disease

Myoglobin, H-FABP and sensitive troponin T were determined in bloodsamples of a total of 234 patients with stable coronary heart disease.The patients did not apparently suffer from an acute coronary event.H-FABP was determined as specified above. Troponin T was determined by ahighly-sensitive troponin T test with a detection limit of 0.002 ng/ml.Patients were subjected to a detailed cardiologic investigationincluding echocardiography and coronary angioplasty. The coronary heartdisease was subclassified into 1-, 2- or 3-vessel diseases, wherebystenosis of more than 50% should occur per vessel. The results are shownin the following tables.

TABLE H-FABP quartiles in patients with documented stable coronary heartdisease. H-FABP [pg/ml] N = 234 1. Quartil 2. Quartil 3. Quartil 4.Quartil N 60 55 59 60 Median H-FABP pg/ml 1132.1 1870.0 2636.5 4086.8Range 0-1550 1565-2208  2223-3337 3357-46370 Age, median 61 64 66 71Coronary artery disease 1-vessel disease 22 9 10 10 2-vessel disease 1510 19 16 3-vessel disease 15 25 23 29 Median NT-proBNP pg/ml 123.0 163.6354.8 835.3 Range 11.2-35802   5.0-5514    6.9-13583  29.6-14953 MedianHs-TnT ng/ml 0.003 0.005 0.007 0.014 Range 0.0-0.113  0.0-0.553 0.0-0.600  0.0-0.708

TABLE Myoglobin quartiles in patients with documented stable coronaryheart disease. Myoglobin [ng/ml] N = 264 Diagnosis Group I: Stablecoronary artery disease 1. Quartil 2. Quartil 3. Quartil 4. QuartilMedian Myoglobin ng/ml 22.87 29.22 37.74 48.84 Range 20.00-25.9125.98-32.73   32.79-42.29 42.50-538.24 Age, median 67 65 67 65 Coronaryartery disease 1-vessel disease 22 20 28 28 2-vessel disease 2 1 4 43-vessel disease 0 0 0 0 Median NT-proBNP pg/ml 154.0 241.8 330.6 606.5Range   17.7-35802 5.0-6431   6.9-14953 27.1-9582  Median Hs-TnT ng/ml0.005 0.005 0.007 0.013 Range  0.0-0.600  0.0-0.553  0.0-0.500 0.0-0.708 Median H-FABP pg/ml 1518 1861 2498 3585 Range   3.5-75240.0-4463  742-6778 1933-46370

1. A method for diagnosing myocardial infarction in a subject, wherein the subject suffers from acute coronary syndrome (ACS) and has a cardiac troponin level which is detectable but lower than the level that is considered indicative for a myocardial infarction (MI), the method comprising: a) obtaining a sample from the subject, b) determining an amount of heart type fatty acid binding protein (H-FABP) in the sample, c) comparing the amount of H-FABP determined to a reference amount of H-FABP for ruling in a recent occurrence of MI and comparing the amount of H-FABP determined to a reference amount of H-FABP for ruling out a recent occurrence of MI, wherein an amount of H-FABP larger than the reference amount for ruling in a recent occurrence of MI is indicative for the recent occurrence of MI and an amount of H-FABP lower than the reference amount for ruling out a recent occurrence of MI is indicative that a MI has not occurred.
 2. The method of claim 1 wherein the reference amount for ruling in a recent occurrence of MI is 5700 pg/ml.
 3. The method of claim 1 wherein the reference amount for ruling out a recent occurrence of MI is 2500 pg/ml.
 4. The method of claim 1, wherein the sample is obtained from the subject within 4 hours after onset of symptoms of ACS.
 5. The method of claim 1 wherein the cardiac troponin is troponin T.
 6. The method of claim 1 wherein the cardiac troponin level ranges from 0.002 ng/ml to 0.1 ng/ml.
 7. A method for diagnosing myocardial infarction in a subject, wherein the subject suffers from acute coronary syndrome (ACS) and has a cardiac troponin level which is detectable but lower than the level that is considered indicative for a myocardial infarction (MI), the method comprising: a) obtaining a sample from the subject, b) determining an amount of heart type fatty acid binding protein (H-FABP) in the sample, c) determining an amount of myoglobin in the sample, d) comparing the amount of myoglobin determined and the amount of H-FABP determined to reference amounts of myoglobin and H-FABP, respectively, for ruling out a recent occurrence of myocardial infarction, and comparing the amount of myoglobin determined and the amount of H-FABP determined to reference amounts of myoglobin and H-FABP, respectively, for ruling in a recent occurrence of myocardial infarction, wherein a myoglobin amount and an H-FABP amount larger than the reference amounts of myoglobin and H-FABP for ruling in a recent occurrence of myocardial infarction is indicative for a recent occurrence of myocardial infarction, and a myoglobin amount and an H-FABP amount lower than the reference amounts of myoglobin and H-FABP for ruling out a recent occurrence of myocardial infarction is indicative that a myocardial infarction has not occurred.
 8. The method of claim 7 wherein the reference amount of myoglobin for ruling in a recent occurrence of myocardial infarction is 77 ng/ml and the reference amount of FABP for ruling in a recent occurrence of myocardial infarction is 5700 pg/ml for H-FABP.
 9. The method of claim 7 wherein the reference amount of myoglobin for ruling out a recent occurrence of myocardial infarction is 55 ng/ml and the reference amount of FABP for ruling out a recent occurrence of myocardial infarction is 2500 pg/ml for H-FABP.
 10. A method for identifying a subject being susceptible to cardiac intervention, wherein the subject suffers from acute coronary syndrome (ACS) and has a cardiac troponin level which is detectable but lower than the level that is considered indicative for a myocardial infarction (MI), the method comprising: a) obtaining a sample from the subject, b) determining an amount of heart type fatty acid binding protein (H-FABP) in the sample. c) comparing the amount of H-FABP determined to a reference amount of H-FABP for ruling in a recent occurrence of MI and comparing the amount of H-FABP determined to a reference amount of H-FABP for ruling out a recent occurrence of MI, wherein an amount of H-FABP larger than the reference amount for ruling in a recent occurrence of MI is indicative for the recent occurrence of MI and an amount of H-FABP lower than the reference amount for ruling out a recent occurrence of MI is indicative that a MI has not occurred, thereby identifying whether the subject is susceptible to cardiac intervention.
 11. A method of deciding on the possible treatment of a subject, wherein the subject suffers from acute coronary syndrome (ACS) and has a cardiac troponin level which is detectable but lower than the level that is considered indicative for a myocardial infarction (MI), the method comprising: a) obtaining a sample from the subject, b) determining an amount of heart type fatty acid binding protein (H-FABP) in the sample, c) comparing the amount of H-FABP determined to a reference amount of H-FABP for ruling in a recent occurrence of MI and comparing the amount of H-FABP determined to a reference amount of H-FABP for ruling out a recent occurrence of MI, wherein an amount of H-FABP larger than the reference amount for ruling in a recent occurrence of MI is indicative for the recent occurrence of MI and an amount of H-FABP lower than the reference amount for ruling out a recent occurrence of MI is indicative that a MI has not occurred, thereby permitting decision on possible treatment of the subject.
 12. A method for ruling out myocardial infarction in a subject, wherein the subject suffers from acute coronary syndrome (ACS) and has a cardiac troponin level which is detectable but lower than the level that is considered indicative for a myocardial infarction (MI), the method comprising: a) obtaining a sample from the subject, b) determining an amount of heart type fatty acid binding protein (H-FABP) in the sample, c) comparing the amount of H-FABP determined to a reference amount of H-FABP for ruling out a recent occurrence of MI, wherein an amount of H-FABP lower than the reference amount for ruling out a recent occurrence of MI is indicative that a MI has not occurred, thereby permitting ruling out myocardial infarction in the subject.
 13. The method of claim 12 wherein said subject already had a cardiac troponin level which is detectable, but lower than the level that is considered as being indicative for a myocardial infarction (MT) at the onset of symptoms of ACS.
 14. A kit for carrying out the method of claim 1 comprising means for determining the amount of H-FABP in a sample of a subject, and, optionally, means for determining the amount of myoglobin in a sample of a subject and means for comparing the amount of H-FABP and, optionally, the amount of myoglobin to at least one reference amount.
 15. The kit of claim 14 further comprising means for determining the amount of a cardiac troponin.
 16. A device for carrying out the method of claim 1 comprising means for determining the amount of H-FABP in a sample of a subject, and, optionally, means for determining the amount of myoglobin in a sample of a subject and means for comparing the amount of H-FABP and, optionally, the amount of myoglobin to at least one reference amount.
 17. The device of claim 16 further comprising means for determining the amount of a cardiac troponin. 